Over the past decade numerous diseases affecting highly active metabolic tissues including the brain and skeletal musculature have been shown to be associated with mutations in the genome of the subcellular organelle, the mitochondria. This organelle is of importance because of its critical role in aerobic metabolism. In addition, a number of maternally inherited diseases have been linked to mutations in the mitochondrial genome, a 16,569 nucleotide pair circular genome. Mendelian, or nuclear, inherited genetic variations and diseases have also been found to affect the mitochondrial function, structure, and genome. Specific regions of the brain appear to be more prone to the occurrence of mitochondrial DNA (mtDNA) deletion mutations. By employing PCR assays to examine brain tissue from 43 age-comparable individuals (between ages 34 and 73), we found a correlation between mitochondrial DNA deletion mutations and conditions associated with chronic hypoxia. In prior studies, utilizing only 6 to 12 clinical samples, mtDNA deletions were reported to increase in specific regions of the brain with aging. However, we found 12 fold and 5 fold higher levels of mtDNA deletions in the putamen and the superior frontal gyrus of the cortex respectively, from age matched individuals who had conditions associated with chronic hypoxia when compared with individuals without evidence of such conditions. These findings suggest that chronic hypoxia should be more closely examined in the pathophysiology of central nervous system diseases. These observations also raise questions as to why these mutations are region specific and whether they are primary or secondary to pathophysiological processes. The regional specificity of the mtDNA deletions may be due in part to variations in regional blood flow, metabolic rates, and the presence of known mutagens, such as nitric oxide. If mtDNA mutation events are primary they may serve as trigger mechanisms for disease processes. The loss of critical mitochondrial functions is particularly detrimental to neurons, which require considerable amounts of energy to restore the transmembrane potentials following each depolarization. The Laboratory is currently initiating animal and tissue culture studies to determine the molecular mechanisms and critical biochemical pathways that may be involved in the occurrence of deletion mutations in the mitochondrial genome. One of the proposed mechanisms for the generation of these deletion mutations involves the action of human mtDNA polymerase (POLG). POLG has been invoked as an active participant in the slip-replication mechanism of human mtDNA deletion mutations. To further our studies concerning the underlying mechanisms of the mtDNA deletions we have localized the POLG gene in man and its homologue, Polg, in mouse. POLG cDNAs were identified by homology with the yeast mtDNA polymerase catalytic subunit (MIP). Fluorescence in situ hybridization (FISH) of human and mouse bacterial artificial chromosomes (BACs), hybridized by the radioactively-labeled POLG cDNAs, mapped to the human chromosome band 15q24-26, and to the mouse chromosome band 13A2, respectively. Sequence analysis by direct sequencing of the BACs has confirmed the localizations. Localization of the mouse homologue (Polg) of the human POLG may facilitate the development of a mouse model for examination of some of the mechanisms of mtDNA mutagenesis. In addition, we are currently adapting the fluorescence in situ hybridization technologies for use in comparative genomic hybridization to search the nuclear genome in CNS regions for amplified or deleted genetic elements. To facilitate our efforts concerning research on the mitochondrial genome the Laboratory has established a database on the World Wide Web, Mitodat (http://www-lecb.ncifcrf.gov/mitoDat/). This database contains: information on proteins that localize to one of the mitochondrial compartments (matrix, inner membrane, inter-membrane space, or outer membrane) and chromosomal locations of genes encoding mitochondrial associated protein, as well as relevant references. In addition, the Laboratory has taken the initiative to establish a special interest mitochondria group for the NIH intramural program. This group meets on a monthly basis to review current research advances.